Evaluation of 6 MALDI-Matrices for 10 ?m Lipid Imaging and On-Tissue MSn with AP-MALDI-Orbitrap

Mass spectrometry imaging is a technique uniquelysuited to localize and identify lipids in a tissue sample. Using anatmospheric pressure (AP-) matrix-assisted laser desorption ioniza-tion (MALDI) source coupled to an Orbitrap Elite, numerous lipidlocations and structures can be determined in high mass resolutionspectra and at cellular spatial resolution, but careful samplepreparation is necessary. We tested 11 protocols on serial brainsections for the commonlyused MALDI matrices CHCA,norharmane, DHB, DHAP, THAP, and DAN in combination withtissue washing and matrix additives to determine the lipid coverage,signal intensity, and spatial resolution achievable with AP-MALDI. Inpositive-ion mode, the most lipids could be detected with CHCA andTHAP, while THAP and DAN without additional treatment offeredthe best signal intensities. In negative-ion mode, DAN showed the best lipid coverage and DHAP performed superiorly forgangliosides. DHB produced intense cholesterol signals in the white matter. One hundredfifty-five lipids were assigned in positive-ion mode (THAP) and 137 in negative-ion mode (DAN), and 76 peaks were identified using on-tissue tandem-MS. The spatialresolution achievable with DAN was 10 mu m, confirmed with on tissue line-scans. This enabled the association of lipid species tosingle neurons in AP-MALDI images. The results show that the performance of AP-MALDI is comparable to vacuum MALDI techniques for lipid imaging